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Background: Stochastic optical reconstruction microscopy (STORM), a super-resolution microscopy technique based on single-molecule localizations, has become popular to characterize sub-diffraction limit targets. However, due to lengthy image acquisition, STORM recordings are prone to sample drift. Existing cross-correlation or fiducial marker-based algorithms allow correcting the drift within each channel, but misalignment between channels remains due to interchannel drift accumulating during sequential channel acquisition. This is a major drawback in multi-color STORM, a technique of utmost importance for the characterization of various biological interactions. Results: We developed RegiSTORM, a software for reducing channel misalignment by accurately registering STORM channels utilizing fiducial markers in the sample. RegiSTORM identifies fiducials from the STORM localization data based on their non-blinking nature and uses them as landmarks for channel registration. We first demonstrated accurate registration on recordings of fiducials only, as evidenced by significantly reduced target registration error with all the tested channel combinations. Next, we validated the performance in a more practically relevant setup on cells multi-stained for tubulin. Finally, we showed that RegiSTORM successfully registers two-color STORM recordings of cargo-loaded lipid nanoparticles without fiducials, demonstrating the broader applicability of this software. Conclusions: The developed RegiSTORM software was demonstrated to be able to accurately register multiple STORM channels and is freely available as open-source (MIT license) at https://github.com/oystein676/RegiSTORM.git and https://doi.org/10.5281/ zenodo.5509861 (archived), and runs as a standalone executable (Windows) or via Python (Mac OS, Linux).

Additional file 1. Supplementary methods, supplementary figures S1-S4, supplementary tables S1-S5.

To assess and describe the aetiology and management of febrile illness in children with primary or acquired immunodeficiency at high risk of serious bacterial infection, as seen in emergency departments in tertiary hospitals. Prospective data on demographics, presenting features, investigations, microbiology, management, and outcome of patients within the ‘Biomarker Validation in HR patients’ database in PERFORM, were analysed. Immunocompromised children (N = 492); 94.3% proven/presumed bacterial (N = 164), 66.1% proven/presumed viral (N = 84), and 93.2% unknown bacterial or viral infections (N = 177). Mortality was 1.9% (N = 11) and 87.1% made full recovery (N = 522). Conclusion: The aetiology of febrile illness in immunocompromised children is diverse. In one-third of cases, no cause for the fever will be identified. Justification for standard intravenous antibiotic treatment for every febrile immunocompromised child is debatable, yet effective. Better clinical decision-making tools and new biomarkers are needed for this population. What is Known:• Immunosuppressed children are at high risk for morbidity and mortality of serious bacterial and viral infection, but often present with fever as only clinical symptom.• Current diagnostic measures in this group are not specific to rule out bacterial infection, and positivity rates of microbiological cultures are low. What is New:• Febrile illness and infectious complications remain a significant cause of mortality and morbidity in HR children, yet management is effective.• The aetiology of febrile illness in immunocompromised children is diverse, and development of pathways for early discharge or cessation of intravenous antibiotics is debatable, and requires better clinical decision-making tools and biomarkers.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

The circadian rhythm generates out-of-equilibrium metabolite oscillations controlled by feedback loops under light/dark cycles. Here we describe a non-equilibrium nanosystem comprising a binary population of enzyme-containing polymersomes capable of light-gated chemical communication, controllable feedback and coupling to macroscopic oscillations. The populations consist of esterase-containing polymersomes functionalized with photo-responsive Donor-Acceptor Stenhouse Adducts (DASA) and light-insensitive semi-permeable urease-loaded polymersomes. The DASA-polymersome membrane becomes permeable under green light, switching on esterase activity and decreasing the pH, which in turn initiates production of alkali in the urease-containing population. A pH-sensitive pigment that absorbs green light when protonated provides a negative feedback loop for deactivating the DASA-polymersomes. Simultaneously, increased alkali production deprotonates the pigment, re-activating esterase activity by opening the membrane gate. We utilise light-mediated fluctuations of pH to perform non-equilibrium communication between the nanoreactors and use the feedback loops to induce work as chemomechanical swelling/deswelling oscillations in a crosslinked hydrogel. We envision possible applications in artificial organelles, protocells, and soft robotics.

Constructing artificial systems that effectively replace or supplement natural biological machinery within cells is one of the fundamental challenges underpinning bioengineering. At the sub-cellular scale, artificial organelles (AOs) have significant potential as long-acting biomedical implants, mimicking native organelles by conducting intracellularly compartmentalized enzymatic actions. The potency of these AOs can be heightened when judiciously combined with genetic engineering, producing highly tailorable biohybrid cellular systems. Here, the authors present a cost-effective, microliter scale (10 µL) polymersome (PSome) synthesis based on polymerization-induced self-assembly for the in situ encapsulation of Gaussia luciferase (GLuc), as a model luminescent enzyme. These GLuc-loaded PSomes present ideal features of AOs including enhanced enzymatic resistance to thermal, proteolytic, and intracellular stresses. To demonstrate their biomodulation potential, the intracellular luminescence of GLuc-loaded PSomes is coupled to optogenetically engineered cardiomyocytes, allowing modulation of cardiac beating frequency through treatment with coelenterazine (CTZ) as the substrate for GLuc. The long-term intracellular stability of the luminescent AOs allows this cardiostimulatory phenomenon to be reinitiated with fresh CTZ even after 7 days in culture. This synergistic combination of organelle-mimicking synthetic materials with genetic engineering is therefore envisioned as a highly universal strategy for the generation of new biohybrid cellular systems displaying unique triggerable properties.

Antibiotic resistance is a severe global health threat and hence demands rapid action to develop novel therapies, including microscale drug delivery systems. Herein, a hierarchical microparticle system is developed to achieve bacteria-activated single- and dual-antibiotic drug delivery for preventing methicillin-resistant Staphylococcus aureus (MRSA) bacterial infections. The designed system is based on a capsosome structure, which consists of a mesoporous silica microparticle coated in alternating layers of oppositely charged polymers and antibiotic-loaded liposomes. The capsosomes are engineered and shown to release their drug payloads in the presence of MRSA toxins controlled by the Agr quorum sensing system. MRSA-activated single drug delivery of vancomycin and synergistic dual delivery of vancomycin together with an antibacterial peptide successfully kills MRSA in vitro. The capability of capsosomes to selectively deliver their cargo in the presence of bacteria, producing a bactericidal effect to protect the host organism, is confirmed in vivo using a Drosophila melanogaster MRSA infection model. Thus, the capsosomes serve as a versatile multidrug, subcompartmentalized microparticle system for preventing antibiotic-resistant bacterial infections, with potential applications to protect wounds or medical device implants from infections.

Additional file 1: Figure S1. (A) Potassium and (B) Magnesium ion release from all glasses in SBF after 1, 2, 4, 6, 24 h, 3, 7, 14 and 21 d. Figure S2. XRD patterns of all four glass compositions before incubation in SBF buffer. Figure S3. TEM-EDS analysis of glass structure of (A) 5Co-A and (B) 2Co-A before incubation in SBF buffer. Scale bar is 200 nm. Figure S4. Representative SEM image of the 2Co-A electrospun composite at low magnification showing the wide variety of fibre diameters. Scale bar is 100 μm.

Theranostics, the combination of diagnosis and therapy, has long held promise as a means to achieving personalised precision cancer treatments. However, despite its potential, theranostics has yet to realise significant clinical translation, largely due the complexity and overriding toxicity concerns of existing theranostic nanoparticle strategies. Here, we present an alternative nanoparticle-free theranostic approach based on simultaneous Raman spectroscopy and photodynamic therapy (PDT) in an integrated clinical platform for cancer theranostics. We detail the compatibility of Raman spectroscopy and PDT for cancer theranostics, whereby Raman spectroscopic diagnosis can be performed on PDT photosensitiser-positive cells and tissues without inadvertent photosensitiser activation/photobleaching or impaired diagnostic capacity. We further demonstrate that our theranostic platform enables in vivo tumour diagnosis, treatment, and post-treatment molecular monitoring in real-time. This system thus achieves effective theranostic performance, providing a promising new avenue towards the clinical realisation of theranostics.

Understanding the mechanisms behind crystal nucleation and growth is a fundamental requirement for the design and production of bespoke nanomaterials with controlled sizes and morphologies. Herein, we select gold (Au) nanoparticles as the model system for our study due to their representative applications in biology, electronics and optoelectronics. We investigate the radiation-induced in situ growth of gold (Au) particles using liquid cell transmission electron microscopy (LCTEM) and study the growth kinetics of non-spherical Au structures. Under controlled electron fluence, liquid flow rate and Au3+ ion supply, we show the favoured diffusion-limited growth of multi-twinned nascent Au seed particles into branched structures when using thin liquid cells (100 nm and 250 nm) in LCTEM, whereas faceted structures (e.g., spheres, rods, and prisms) formed when using a 1 μm thick liquid cell. In addition, we observed that anisotropic Au growth could be modulated by Au-binding amyloid fibrils, which we ascribe to their capability to regulate Au3+ ion diffusion and mass transfer in solution. We anticipate that this study will provide new perspectives on the shape-controlled synthesis of anisotropic metallic nanomaterials using LCTEM.

Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.

This study explored the regenerative osteogenic response in the distal femur of sheep using scaffolds having stiffness values within, and above and below, the range of trabecular bone apparent modulus. Scaffolds 3D-printed from stiff titanium and compliant polyamide were implanted into a cylindrical metaphyseal defect 15 × 15 mm. After six weeks, bone ingrowth varied between 7 and 21% of the scaffold pore volume and this was generally inversely proportional to scaffold stiffness. The individual reparative response considerably varied among the animals, which could be divided into weak and strong responders. Notably, bone regeneration specifically within the interior of the scaffold was inversely proportional to scaffold stiffness and was strain-driven in strongly-responding animals. Conversely, bone regeneration at the periphery of the defect was injury-driven and equal in all scaffolds and in all strongly- and weakly-responding animals. The observation of the strain-driven response in some, but not all, animals highlights that scaffold compliance is desirable for triggering host bone regeneration, but scaffold permanence is important for the load-bearing, structural role of the bone-replacing device. Indeed, scaffolds may benefit from being nonresorbable and mechanically reliable for those unforeseeable cases of weakly responding recipients.

Acoustic patterning using ultrasound standing waves has recently emerged as a potent biotechnology enabling the remote generation of ordered cell systems. This capability has opened up exciting opportunities, for example, in guiding the development of organoid cultures or the organization of complex tissues. The success of these studies is often contingent on the formation of tightly-packed and uniform cell arrays; however, a number of factors can act to disrupt or prevent acoustic patterning. Yet, to the best of our knowledge, there has been no comprehensive assessment of the quality of acoustically-patterned cell populations. In this report we use a mathematical approach, known as Voronoï tessellation, to generate a series of metrics that can be used to measure the effect of cell concentration, pressure amplitude, ultrasound frequency and biomaterial viscosity upon the quality of acoustically-patterned cell systems. Moreover, we extend this approach towards the characterization of spatiotemporal processes, namely, the acoustic patterning of cell suspensions and the migration of patterned, adherent cell clusters. This strategy is simple, unbiased and highly informative, and we anticipate that the methods described here will provide a systematic framework for all stages of acoustic patterning, including the robust quality control of devices, statistical comparison of patterning conditions, the quantitative exploration of parameter limits and the ability to track patterned tissue formation over time.

Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120–150 µm and a spacing of 180–220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics.

Neural tissue engineering (TE) represents a promising new avenue of therapy to support nerve recovery and regeneration. To recreate the complex environment in which neurons develop and mature, the ideal biomaterials for neural TE require a number of properties and capabilities including the appropriate biochemical and physical cues to adsorb and release specific growth factors. Here, we present neural TE constructs based on electrospun serum albumin (SA) fibrous scaffolds. We doped our SA scaffolds with an iron-containing porphyrin, hemin, to confer conductivity, and then functionalized them with different recombinant proteins and growth factors to ensure cell attachment and proliferation. We demonstrated the potential for these constructs combining topographical, biochemical, and electrical stimuli by testing them with clinically relevant neural populations derived from human induced pluripotent stem cells (hiPSCs). Our scaffolds could support the attachment, proliferation, and neuronal differentiation of hiPSC-derived neural stem cells (NSCs), and were also able to incorporate active growth factors and release them over time, which modified the behavior of cultured cells and substituted the need for growth factor supplementation by media change. Electrical stimulation on the doped SA scaffold positively influenced the maturation of neuronal populations, with neurons exhibiting more branched neurites compared to controls. Through promotion of cell proliferation, differentiation, and neurite branching of hiPSC-derived NSCs, these conductive SA fibrous scaffolds are of broad application in nerve regeneration strategies.

Analyzing lipid composition and distribution within the brain is important to study white matter pathologies that present focal demyelination lesions, such as multiple sclerosis. Some lesions can endogenously re-form myelin sheaths. Therapies aim to enhance this repair process in order to reduce neurodegeneration and disability progression in patients. In this context, a lipidomic analysis providing both precise molecular classification and well-defined localization is crucial to detect changes in myelin lipid content. Here we develop a correlated heterospectral lipidomic (HSL) approach based on coregistered Raman spectroscopy, desorption electrospray ionization mass spectrometry (DESI-MS), and immunofluorescence imaging. We employ HSL to study the structural and compositional lipid profile of demyelination and remyelination in an induced focal demyelination mouse model and in multiple sclerosis lesions from patients ex vivo. Pixelwise coregistration of Raman spectroscopy and DESI-MS imaging generated a heterospectral map used to interrelate biomolecular structure and composition of myelin. Multivariate regression analysis enabled Raman-based assessment of highly specific lipid subtypes in complex tissue for the first time. This method revealed the temporal dynamics of remyelination and provided the first indication that newly formed myelin has a different lipid composition compared to normal myelin. HSL enables detailed molecular myelin characterization that can substantially improve upon the current understanding of remyelination in multiple sclerosis and provides a strategy to assess remyelination treatments in animal models., A novel heterospectral analysis technique combining Raman spectroscopy and DESI mass spectrometry allows uncovering of lipid compositional differences during remyelination in multiple sclerosis.

Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. We anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

The normal structure of human feet is optimized for shock dampening during walking and running. Foot binding was a historical practice in China aimed at restricting the growth of female feet for aesthetic reasons. In a bound foot the shock‐dampening function normally facilitated by the foot arches is withdrawn, resulting in the foot functioning as a rigid extension of the lower leg. An interesting question inspiring this study regards the nature of adaptation of the heel bone to this nonphysiological function using the parameters of cancellous bone anisotropy and 3D fabric topology and a novel intertrabecular angle (ITA) analysis. We found that the trabecular microarchitecture of the normal heel bone, but not of the bound foot, adapts to function by increased anisotropy and preferred orientation of trabeculae. The anisotropic texture in the normal heel bone consistently follows the physiological stress trajectories. However, in the bound foot heel bone the characteristic anisotropy pattern fails to develop, reflecting the lack of a normal biomechanical input. Moreover, the basic topological blueprint of cancellous bone investigated by the ITA method is nearly invariant in both normal and bound foot. These findings suggest that the anisotropic cancellous bone texture is an acquired characteristic that reflects recurrent loading conditions; conversely, an inadequate biomechanical input precludes the formation of anisotropic texture. This opens a long‐sought‐after possibility to reconstruct bone function from its form. The conserved topological parameters characterize the generic 3D fabric of cancellous bone, which is to a large extent independent of its adaptation to recurrent loading and perhaps determines the mechanical competence of trabecular bone regardless of its functional adaptation. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Amyloid fibrillation is a nucleation-dependent process known be involved in the development of more than 20 progressive and chronic diseases. The detection of amyloid formation at the nucleation stage can greatly advance early diagnoses and treatment of diseases. In this work, we developed a new assay for the early detection of amylin fibrillation using the biarsenical dye 4,5-bis(1,3,2-dithiarsolan-2-yl)fluorescein (FlAsH), which could recognise tetracysteine motifs and transform from non-fluorescent form into strongly fluorescent complexes. Due to the close proximity of two cysteine residues within the hydrophilic domain of amylin, a non-contiguous tetracysteine motif can form upon amylin dimerisation or oligomerisation, which can be recognised by FlAsH and emit strong fluorescence. This enables us to report the nucleation-growth process of amylin without modification of the protein sequence. We showed that the use of this assay not only allowed the tracking of initial nucleation events, but also enabled imaging of amyloid fibrils and investigation of the effects of amyloid inhibitor/modulator toward amylin fibrillation.

Research data supporting the publication: Kallepitis, C. et al. Quantitative volumetric Raman imaging of three dimensional cell cultures. Nat. Commun. 8, 14843 doi: 10.1038/ncomms14843 (2017).

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets., UK Regenerative Medicine Platform Hubs ‘Acellular Approaches for Therapeutic Delivery’(MR/K026682/1) and ‘A Hub for Engineering and Exploiting the Stem Cell Niche’ (MR/K026666/1). M.M.S. also acknowledges the grant ‘State of the Art Biomaterials Development and Characterization of the Cell-Biomaterial Interface’ (MR/L012677/1) from the Medical Research Council. J.-P.S. and M.M.S. were also supported by the Medical Engineering Solutions in the Osteoarthritis Centre of Excellence, funded by the Wellcome Trust and the Engineering and Physical Sciences Research Council (EPSRC) (088844).

Nitric oxide (NO) has been shown to lower intraocular pressure (IOP), however its therapeutic effects on outflow physiology are location- and dose-dependent. Here, a NO delivery platform that directly targets the resistance-generating region of the conventional outflow pathway and locally liberates a controlled dose of NO is reported. An increase in outflow facility (decrease in IOP) is demonstrated in mouse model.

Antibiotic resistance (ABR) is a serious global health problem necessitating new bactericidal approaches as well as biosensors which can detect bacterial infection. Vesicular nanostructures have shown widespread promise for numerous biomedical applications in drug delivery and biosensing. A recently reported vesicle class composed of amphiphilic Janus dendrimers (AJDs), termed dendrimersomes (DSs), represent a promising system marrying the diverse chemistry of polymers and exact molecular definition of lipids. However, DSs have not yet been explored in depth for potential biomedical applications and, as yet, have not been used to construct nanoreactors – nanocompartments which encapsulate enzymes and permit reactions within a confined and localised space. Nanoreactors are of interest for myriad biomedical applications, some of which have been inspired by natural organelles. Herein, synthesis of a specific AJD was conducted and DS self-assembly investigated using various characterisation techniques revealing key properties of the DSs, particularly, their inherently semipermeable membranes, an essential property for use as a nanoreactor. Consequently, and inspired by the microbicidal function of the neutrophil phagosome, a DS-based nanoreactor was developed for an antibiotic-free bactericidal application. This was achieved by encapsulation of glucose oxidase and myeloperoxidase within DSs, enabling localised conversion of glucose to the highly potent microbicide, hypochlorite. This nanoreactor exerted a potent bactericidal effect against two bacterial pathogens on the WHO list for which new antibiotics are needed. Furthermore, a proof-of-concept is presented for harnessing the activity of membrane-lytic bacterial toxins to activate the nanoreactors through glucose release from separate lipid vesicles. Overall, the repertoire of potential DS biomedical applications has been expanded by this thesis and sets the basis for future nanoreactor applications. Lastly, again inspired by the activity of bacteria toxins, a liposome-based biosensor is presented towards the in vivo detection of Staphylococcus aureus lung infection using a colorimetric urinary readout. This system is composed of a liposome encapsulating renally clearable gold nanoclusters (AuNCs) with peroxidase-like activity. AuNC loading and in vitro release upon enzyme and toxin incubation was achieved and initial in vivo experiments performed to assess the utility of the system for detecting lung infection. Furthermore, initial work towards imparting an extra logic gate into the AuNC-liposome sensor for more specific disease detection is discussed. Overall, this thesis presents two vesicle-based systems towards treatment and detection of bacterial infection, which are in urgent need of alternative strategies due to the spread of ABR. Open Access

Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease and cancer. Improving localised delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Employing immune cells for active transport of drugs and drug-loaded nanocarriers to a target site is a promising recent approach. Particularly, in the context of drug delivery for anti-inflammation therapy, immune cells have the intrinsic functions to migrate to and infiltrate the inflamed tissue. Here, a delivery strategy using neutrophils loaded with methotrexate (MTX)-liposomes ex vivo to deliver MTX to target sites was first investigated in vitro. This hybrid system efficiently migrated following an inflammatory chemokine gradient and triggered release of loaded liposomes via neutrophil extracellular traps (NETs) formation in an inflammatory environment was achieved. Subsequent re-uptake of the released liposomes by target macrophages provided detailed support for in vivo treatment studies. In parallel, macrophages were evaluated as alternative immune cells to carry nanoparticles. Given the advantage of a well-defined release mechanism (NETs formation), MTX-liposome loaded neutrophils were chosen over a macrophage-based system to be further validated in vivo using two mouse models of inflammation, a lipopolysaccharide (LPS)-injury skeletal muscle model and a myocardial ischemia reperfusion injury (IRI) model. The migratory behaviour of liposome-loaded neutrophils was confirmed by demonstrating the neutrophil-mediated delivery of liposomes to the inflamed muscle and the injured heart. A single low-dose injection of the hybrid system locally reduced inflammatory cytokine levels in the inflamed muscle and slightly improved the pumping efficiency of the IRI heart. These results highlight the advantages of immune cell-mediated drug delivery, which is a versatile strategy that allows combinations with different types of nanoparticles encapsulating various drugs to reduce tissue inflammation and actively promote repair in inflammatory diseases. Open Access

The recent ExecutiveOrder by President Trump attempting to ban temporarily the citizens of seven countries (Iran, Iraq, Libya, Somalia, Sudan, Syria, and Yemen) from entering the United States is having significant consequences within the country and around the world. The Order poses a threat to the health and vitality of science, barring students and scientists from these countries from traveling to the United States to study or to attend conferences. In preventing those members of the international scientific community from traveling beyond U.S. borders without guaranteed safe return, the Executive Order demeans them; in so doing, it demeans us all. Universities and research communities are especially impacted, as major universities have students and often faculty holding passports from one of these seven countries. This temporary ban would affect refugees fleeing war-torn areas, challenging the long-standing notion that the United States is a safe haven for those fleeing persecution and war in addition to being a magnet for talent from every corner of the world. The pages of this journal reflect the geographic, ethnic, and cultural diversity that underpins great science. The ban impacts domestic and global scientific efforts and communities. Science succeeds through the cooperation between collections of individuals and teams around the world discovering and learning from each other. To ensure rapid scientific progress, open communication and exchange between scientists are essential. As scientists, engineers, and clinicians, we have benefited from open interactions and collaborations with visitors and students from all parts of the world as well as through scientific publications and discussions at scientific meetings.

Stereolithography printing stands out as a promising additive manufacturing technique for the production of bone graft substitutes and other tissue-engineered constructs due to a high spatial resolution, generation of internal microstructures, and high productivity. A current obstacle for the application of this manufacturing method is the limited library of biocompatible materials and non-standard characterization methods. In this work, a low molecular weight polycaprolactone (PCL) triacrylate resin was synthesized for stereolithography printing, characterized using ISO standard testing methods, and printed into complex lattice structures. The mechanical properties of the resin were characterized in tension (E = 12.2 MPa, σbreak = 1.49 MPa, εbreak = 13 %) and in compression (E = 97.99 MPa, σmax = 21.1 MPa, εmax = 30.1 %). Furthermore, the trade-off between scaffold stiffness and porosity was investigated for two cellular lattice structures (octet-truss and tetradecahedron) in silico using Finite Element Analysis (FEA). Initial FEA results indicate that the octet-truss lattice exhibits a higher stiffness at a lower relative density (ρ’ < 20 %) than the tetradecahedron lattice. Open Access

Inappropriate wound healing represents a considerable medical challenge associated with high mortality. However, improving on current wound healing therapies has proven difficult due to the complex and dynamic wound environment. The complexity of the wound healing process also puts high demands on the animal models used in wound research, since ideally such models should encompass the full complexity of the wound healing process, and at the same time be accessible for advanced biomedical analysis methods. In this thesis, the aim was to further develop the use of zebrafish embryos in wound healing research. Key advantages of zebrafish embryo models are the ability to visualize complex biological processes in high detail in intact tissues, as well as highly tractable genetics. The first part of the work describes the development of a zebrafish embryo model for investigating the immunomodulatory properties of hydrogels derived from decellularized extracellular matrix (ECM). The results demonstrate that the hydrogels can be properly injected into the embryos and that the host-materials interactions can be explored in detail inside live zebrafish embryos during wound healing. This constitutes a new in vivo model for investigating immunomodulatory materials in a realistic wound healing context. The second part of the work describes the development of a confocal Raman spectrometry imaging (cRSI) method for biomolecular characterization and the study of biological processes in zebrafish. This represents a new imaging modality that enables simultaneous inspection of a multitude of biomolecules in a label-free manner. The use of cRSI was demonstrated for biomolecular discrimination of mycobacteria in a zebrafish infection model, and for live in vivo imaging of zebrafish during the early wound response. Taken together, the work in this thesis has provided a new methodologies and insight for the use in zebrafish embryo models in wound healing research. Open Access

The design of materials able to undergo changes in response to an applied stimulus (e.g. temperature, pH or magnetic fields) is relevant for biomedical applications. In the context of hydrogels, the design of triggers for hydrogelation has enabled precise control over hydrogelation kinetics and mechanical properties. One trigger that has yet to be explored for hydrogelation is ultrasound; a widely-used biomedical platform that is non-invasive, with tuneable tissue penetration depth and high spatiotemporal control. The use of ultrasound as a remote trigger for enzymatic activity and hydrogelation was explored in this thesis. In particular, the ability of liposomes and microbubble-liposome conjugates to release encapsulated payloads upon ultrasound exposure was leveraged. The designed field-responsive system required that the amount of encapsulated calcium in liposomes was maximised. Hence, cryo-TEM and small-angle neutron scattering were used to investigate the effect of the formulation method and the lipid composition on vesicle lamellarity, which determines the volume of the internal liposomal aqueous compartment. In another study, X-ray and neutron scattering corroborated with all-atom molecular dynamics simulations were used to elucidate the effect of sodium and calcium ions on ethanol-induced lipid membrane interdigitation. The results of this study, which spanned a wide range of length scales, furthered the understanding of ethanol-induced interdigitation of bulk and vesicular lipid formulations, with important implications for the production of interdigitation-fusion vesicles. Calcium-loaded liposomes produced via the interdigitation fusion vesicle method that were able to release their payload upon ultrasound exposure were utilised to trigger the catalytic activity of a calcium-dependent tissue transglutaminase. The ultrasound-activated transglutaminase could then catalyse intermolecular covalent crosslinking between the lysine and glutamine sidechain residues of soluble fibrinogen molecules, yielding fibrinogen hydrogels. Precise control over these processes could be achieved, with the calcium release, catalysis rate and hydrogelation rate all shown to be dependent upon the ultrasound exposure time. Calcium-loaded liposomes were also conjugated to the surface of gaseous microbubbles that are commonly used for in vivo drug delivery. These microbubble-liposome conjugates exhibited enhanced response to the applied acoustic field and could also be used for ultrasound-triggered hydrogelation. Taken together, these results represent an entirely new class of stimuli for enzyme activity and hydrogelation and open up a wide range of opportunities for ultrasound-triggered molecular biology, synthetic biology and material science. Open Access

Cardiovascular diseases are the primary cause of death globally, responsible for approximately 17.5 million deaths per year. In particular, myocardial infarction caused by the obstruction of a coronary artery can lead to heart failure. A growing number of studies have demonstrated that extracellular vesicles (EVs) exhibit great potential as cell-free cardioprotective agents, which could overcome many of the technical, ethical and regulatory hurdles associated with cell-based therapies. However, the beneficial effects of administered EVs are short-lived; a major limitation that could be circumvented using a material-based approach for increased retention and sustained release. Therefore, the main objective of this study was to develop a translational approach for repairing the infarcted heart, in the form of injectable, ‘off-the-shelf’ EV-loaded synthetic microgels. This platform was designed to improve EV stability and retention time at the site of administration, with sustained and environmentally-responsive release of EVs to the surrounding damaged tissue. Methods for fabricating monodisperse environmentally-sensitive microgels using a microfluidic-based platform were developed, and a first proof-of-concept of the feasibility of loading and releasing EVs from the microgels was demonstrated in vitro and in vivo. Open Access

Nanomedicine represents a challenging and highly multidisciplinary research field, concerned with the development and study of nanoformulations for diagnostic and/or therapeutic purposes. The nano-sized particles of interest are increasingly complex. Their translational potential has been hampered by difficulties in their thorough characterisation. On the nano scale, small variations in size and composition can have large implications for their pharmacodynamic and pharmacokinetic behaviour. More precise techniques are therefore required to address these challenges. This thesis describes novel, advanced characterisation methods designed for the detailed study of single nanoparticles and their interaction and uptake behaviour with cells. A platform technology for Single Particle Automated Raman Trapping Analysis – SPARTA - was developed, capable of non-destructive, label-free and automated comprehensive single particle analysis. With the SPARTA system, the composition, functionalisation, size and dynamic reactions on the surface can be investigated in detail, of a wide variety of nanoparticles, through their Raman spectra. A further improved, custom designed SPARTA 2.0 platform was built, optimised for the analysis of complex biological particles, such as EVs. EVs represent a high potential as biomarkers, studied here in the context of breast cancer. The SPARTA 2.0 platform was able to resolve compositional differences between non-cancerous and cancer cell-derived EVs with excellent sensitivity and specificity. This highlights the possibility for development of new minimally invasive diagnostic approaches. In addition, a new imaging strategy for investigation of the EV-cellular interaction is presented, based on 3D Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM). FIB-SEM allows the generation of 3D models of the subcellular structure and visualisation of the cellular trafficking of nanoparticles. This represents a powerful new approach for investigating EV uptake. The methods developed in this thesis allow for the single particle-based analysis of a wide variety of nanoformulations and EVs, to aid in understanding their composition, applicability and cellular interactions. Open Access

Myocardial infarction (MI) is characterised by the irreversible death of cardiac muscle with loss of up to 1 billion cardiomyocytes (CM). Despite survival post-MI dramatically improving in the last two decades, more than 20% of patients suffering MI will still develop heart failure (HF), an incurable condition where the heart is no longer able to meet the body’s needs for blood supply. Amongst novel therapeutic avenues currently being explored, intramyocardial delivery of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) holds great promise to replace the lost functional tissue. However, the effects of the ischemic microenvironment on these cells still need to be investigated, and protective strategies need to be developed. This thesis examines the delivery of the pro-survival growth factor Insulin like Growth Factor-1 (IGF-1) in the settings of hiPSC-CMs exposed to acidic pH and through a hydrogel-based approach in an in vivo model of MI. Following MI, the heart switches from aerobic metabolism to anaerobic glycolysis, causing a pH drop to 6.5-6.8. The aim of the first part of this thesis was to mitigate the effects of acidic pH on hiPSC-CMs using the pro-survival growth factor IGF-1. It was shown that acidic pH negatively affects hiPSC-CMs in terms of viability, metabolic activity, cardiac gene expression and CMs yield obtained through differentiation. IGF-1 was able to recover the effects of acidic pH, and it could, therefore, be used as a protective strategy for in vivo cell therapy approaches. Another promising strategy for preventing HF progression following MI is the minimally invasive delivery of injectable hydrogels, which can provide mechanical support to damaged tissue and deliver bioactive factors with pro-survival actions. Here, a thermoresponsive injectable hydrogel composed of a triblock copolymer of polyethylene glycol (PEG) and polycaprolactone (PCL) was synthesised and characterised in vitro and in vivo. The hydrogel was prepared with or without insulin-like growth factor-1 (IGF-1) and injected intramyocardially in a mouse MI model. Echocardiography, strain analysis and histological assessments showed that the injection of the biodegradable thermoresponsive hydrogel was effective in ameliorating pathological remodelling, improving overall cardiac function and myocardial mechanics. In the future, implementing novel therapeutic approaches like the ones presented in this thesis could prevent the progression to HF, improving the quality of life of patients affected by myocardial infarction and limiting the socio-economic burden of the disease. Open Access

This thesis presents three multimodal optical (light-based) systems designed to improve the capabilities of existing optical modalities for cancer diagnostics and theranostics. Optical diagnostic and therapeutic modalities have seen tremendous success in improving the detection, monitoring, and treatment of cancer. For example, optical spectroscopies can accurately distinguish between healthy and diseased tissues, fluorescence imaging can light up tumours for surgical guidance, and laser systems can treat many epithelial cancers. However, despite these advances, prognoses for many cancers remain poor, positive margin rates following resection remain high, and visual inspection and palpation remain crucial for tumour detection. The synergistic combination of multiple optical modalities, as presented here, offers a promising solution. The first multimodal optical system (Chapter 3) combines Raman spectroscopic diagnostics with photodynamic therapy using a custom-built multimodal optical probe. Crucially, this system demonstrates the feasibility of nanoparticle-free theranostics, which could simplify the clinical translation of cancer theranostic systems without sacrificing diagnostic or therapeutic benefit. The second system (Chapter 4) applies computer vision to Raman spectroscopic diagnostics to achieve spatial spectroscopic diagnostics. It provides an augmented reality display of the surgical field-of-view, overlaying spatially co-registered spectroscopic diagnoses onto imaging data. This enables the translation of Raman spectroscopy from a 1D technique to a 2D diagnostic modality and overcomes the trade-off between diagnostic accuracy and field-of-view that has limited optical systems to date. The final system (Chapter 5) integrates fluorescence imaging and Raman spectroscopy for fluorescence-guided spatial spectroscopic diagnostics. This facilitates macroscopic tumour identification to guide accurate spectroscopic margin delineation, enabling the spectroscopic examination of suspicious lesions across large tissue areas. Together, these multimodal optical systems demonstrate that the integration of multiple optical modalities has potential to improve patient outcomes through enhanced tumour detection and precision-targeted therapies. Open Access

Extracellular Vesicles (EV) are a complex collection of cell derived particles which efficiently transport functionally active bio-molecules over large inter-cellular distances and across biological barriers. To realise the potential of EV to act as next-generation personalised drug delivery vectors novel methods are needed to engineer EV contents and to characterise the interactions of EV with various cellular targets. The rapid pace of development of EV for therapeutic delivery means that the need for robust and sensitive methods which accurately elucidate EV-cell interactions are ever increasingly required. In this work, EV are engineered to carry a small molecule transgene activator called doxycycline (DXC). Dosing of DXC-sensitive reporter cells with loaded DXC-EV was used to trigger red fluorescent protein (RFP) expression in recipient cells. This response was spatiotemporally monitored and the dynamics and penetration of DXC-EV delivery within reporter cell systems cultured as mono-layers, as well as, more advanced 3D multi-cellular spheroid models overtime was tracked overtime. In addition, this reporter system was applied to investigate to study whether EV interactions with target cells could be modulated through the application of ultrasound to release EV from microbubble-EV complexes. Ultrasound was applied in this context to gain remote physical control over EV delivery and overcome diffusional barriers faced when EV need to signal to cell culture systems embedded within 3D collagen micro-environments. Overall, this thesis will examine cell engineering and post-harvest EV modification in order to engineer EV for enhanced therapeutic loading, and, will devise novel biomaterial inspired approaches to manipulate and gain physical control over EV to cell communication within tissue engineering scaffolds. Overall, these advances could be applied in subsequent studies to enhance the therapeutic properties of EV in the context of regenerative medicine. Open Access

Cardiac tissue engineering to replace damaged areas of the postmitotic heart is still presented with significant challenges, due to the complex and dynamic interplay of electrical, mechanical and biochemical signals involved in the myocardium. The advancement of regenerative approaches is focussed on understanding the underlying regulatory mechanisms involved throughout cardiac development. However, current knowledge of how biophysical cues in the stem cell niche can modulate cell behaviour is limited. Firstly, polyacrylamide-co-acrylic acid was used as an in vitro stiffness-tuneable platform to test the effect of substrate mechanics on human induced pluripotent stem cell (hiPSC) differentiation into cardiomyocytes (CM). The results showed that the optimum differentiation efficiency level peaked at the embryonic-like stiffness of 560 Pa, with increased upregulation of cardiac genes. Functionally, hiPSC-CMs showed a biphasic relationship with a faster calcium transient and higher force generation at cardiac physiological stiffness. Next, shape was incorporated into the experimental design via CardioArray, a custom-built platform which mimics both the stiffness and shape of an adult human CM. This system can accommodate individual hiPSC-CMs to adopt the 3D geometry of an adult CM, while at the same time providing the relevant stiffness cues from the underlying substrate. The results highlighted the specific contribution of stiffness and 3D shape to α-sarcomeric structure, cell membrane stiffness, single cell gene expression and intracellular calcium cycling. Finally, the electrical microenvironment was investigated as a third infleuncing factor on hiPSC-CM development. A hybrid conductive polyaniline-Scl2 scaffold was fabricated, showing long term electronic stability and no cell toxicity when interfaced with electrosensitive hiPSC-CMs. This could provide electromechanical stability in model studies. Improvement of conduction velocity was observed in an in vitro myocardial slice model. As a whole, this thesis demonstrates the differential effects of substrate mechanics on hiPSC cardiac differentiation, providing a novel crucial understanding of how biophysical cues modulate the stem cell niche during differentiation and in vitro culture. Open Access

Tissue engineering research has opened a new chapter in modern medicine since it emerged as a mainstream research field in the early 90s. Until now, however, effective strategies to fully emulate the complexity of natural tissue remain elusive. One of the key features in the development of complex tissue structures is the presence of morphogen gradients during development. In nature, from squid beaks to human teeth, gradients are preserved in many structures after evolution. In a living organism, gradients play an essential role in defining the physiological function. The formation of these gradients is often largely dictated by an anisotropic distribution of different morphogens present during development. The spatial difference in concentration of different morphogens results a spatial variance in cell signalling, patterning the development of tissue and leading to the formation of heterogeneous structure. Although these principles of development are well-established, the overwhelming majority of in vitro engineering strategies use uniform scaffolds and spatially invariant growth factor delivery to produce homogeneous tissue constructs. It is clear that more sophisticated fabrication processes are required to replicate the native complexity and fulfil the functional requirements of tissue grafts. A few material strategies have been developed that can heterogeneously deliver biological or mechanical cues; however, most of them are limited by complex fabrication procedures or restricted compatibility with different material systems. By establishing signaling factor gradients within tissue engineering scaffolds, the formation of heterogeneous tissue interfaces can be achieved. This thesis will demonstrate two gradient casting strategies to emulate physiological gradients, exploiting magnetism and buoyancy to distribute growth factors. These strategies are shown to be capable of establishing gradients in different materials, and are used to engineer osteochondral tissue. The strategies proposed in this research are designed to be widely applicable and easy to reproduce. It is hoped that these strategies may be adapted and tailored for wider use by the tissue engineering field, allowing development of complex, functional tissue by mimicking the processes used by nature. Open Access

This thesis describes the optimisation of the fabrication protocols for vertically-aligned nanostructures to be interfaced with living cells. The dissertation is organised around four aims. The first aim describes the optimisation of a nanoneedle fabrication process. Nanoneedles are regular arrays of conical mesoporous-silicon nanostructures, recently developed by the Stevens Group. The second aim describes one potential novel development for nanoneedles, in the field of biosensing. This involves the selective deposition of silver nanoparticles at the nanoneedle tips, in order to take advantage of Raman-spectroscopy-signal-enhancement phenomena. These were envisioned to facilitate the detection of Raman-active molecules, as well as the investigation of cellular processes. The third aim represents another direction in broadening the horizon of the nanoneedle applications, in the field of cell transfection. This concerns the fabrication of interdigitated, planar electrodes, having nanoneedle-bearing digits. This way, a portable device was envisioned, which could exploit tip-field-enhanced electroporation, as well as tight membrane interface, for improved delivery of molecules to cells. The fourth aim describes the optimisation of the fabrication of nanowires. These were interfaced with bacteria, for prokaryotic-cell transformation purposes. This project is conceptually similar to the nanoneedle-mediated delivery of molecules that has been proven in the literature, but on a smaller scale. Bacteria cells are indeed smaller that eukaryotic ones, thus smaller nanostructures were developed to deliver cargoes into them. This thesis aims to give a contribution to the field of vertically-aligned nanostructures for nanomedicine. This field needs further insights before devices can be used in clinical settings but holds great promises for the future. Open Access